Over 200,000 workers are directly employed in the production and use of diisocyanates, worldwide. Potential exposures to diisocyanates occur in virtually all aspects of our lives from agriculture to transport to leisure. They are commonly used in paints, glues/binders and foams.
Diisocyanates (dNCOs) are used in the production of polyurethanes such as polyurethane foams, elastomers and coatings13. Monoisocyanates (one N═C═O/molecule) are used in nonpolymer applications such as the production of insecticides, pesticides and herbicides14. The most widely used compounds are diisocyanates, which contain two isocyanate groups, and polyisocyanates, which are usually derived from diisocyanates and may contain several isocyanate groups. The most common monomeric dNCOs are toluene diisocyanate (TDI), methylene diphenyl diisocyanate (MDI) and hexamethylene diisocyanate (HDI). Others include naphthalene diisocyanate (NDI), methylene bis-cyclohexylisocyanate (HMDI) (hydrogenated MDI), and isophorone diisocyanate (IPDI). Examples of widely used polyisocyanates include HDI biuret and HDI isocyanurate.
Toluene diisocyanates are reactive intermediates often used to form urethane links. For example, toluene diisocyanates are used in combination with polyether and polyester polyols to produce polyurethane products. TDI is available in commercial preparations which often include an 80:20 mixture of 2,4-TDI and 2,6-TDI15.
Workers are exposed to monomeric and polymeric forms of diisocyanates. The main route of exposure to dNCOs is through inhalation. Other common routes of exposure include eye contamination and skin contamination16.
Responses to dNCOs exposure vary widely from mild irritation of the airways to more severe effects, including bronchospasm. Isocyanates are powerful irritants to the mucous membranes of the eyes, gastrointestinal tract and respiratory system17. Direct skin contact can cause marked dermal inflammation.
Respiratory effects are a primary toxicological manifestation of repeated exposure to diisocyanates16. Exposure to dNCOs are the most commonly reported cause of occupational asthma1.
Diisocyanates can also sensitize workers, making them subject to allergic rhinitis, allergic contact dermatitis and asthma attacks upon re-exposure. Death from severe asthma in sensitized subjects has been reported1.
Due to their widespread use in the general population particularly from products commercially available at hardware stores actual rates of dNCO induced diseases may be under-reported. Health aspects of diisocyanates exposure have been subjected to intensive research, in terms of both human and animal toxicological studies. Dose-dependent responses to higher levels of dNCOs include respiratory, dermal and mucous membrane irritation. Hypersensitivity reactions to dNCO's include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis.
Diisocyanates can bind to proteins through a number of chemical functional groups and the nature of this binding may depend on the protein and the local environment. FIG. 1 illustrates reactions of isocyanates with a biomolecule, such as protein, to form conjugates.
Conjugation (haptenation) of diisocyanate to human proteins after exposure is commonly accepted as an important primary event in the development of diisocyanate-induced allergic sensitization and asthma. Diisocyanates have been shown to bind to skin and lung resident proteins. The major adducts found in the blood are conjugates to hemoglobin and albumin5. TDI-conjugated lung proteins were co-localized with keratin, tubulin, laminin and actin2-11, 19. It is believed, that TDI binding, in vivo, demonstrates selectivity with respect to the target proteins. Detection of dNCO exposure and diagnosis of related pathological conditions is difficult. For example, detection of dNCO exposure-induced antibodies against dNCO in a subject is challenging and is typically attempted using poorly characterized haptenated albumin.
While asthma is considered an inflammatory disorder of the conducting airways, it is becoming increasingly apparent that the disease is heterogeneous with respect to immunopathology17. TDI-specific IgE can be detected in only about 20% of the TDI-asthmatics, suggesting that immunological pathways other than Type I allergic mechanisms, may predominate in the majority of the asthmatics. Although the role of specific IgE antibody has been investigated, the results thus far point to discrepancies or rather low associations between specific IgE antibodies and disease24-28.
The contribution of using inappropriate antigen to the lack of specific-IgE detection in dNCO asthmatics is not known, but most studies evaluating different haptenated protein preparations, usually find differences in affinities of anti-TDI IgEs, but rarely identify a significant increase in TDI specific-IgE prevalence in TDI asthmatics.
The short circulating half-life of unbound serum IgE of about 2 days may be of unique importance to occupational illnesses such as isocyanate asthma. Brief periods away from the workplace may result in a decrease in serum IgE levels to levels undetected by conventional methods29. Without accurate exposure information, negative isocyanate-specific IgE assays may lead to misdiagnosis and false conclusions about pathogenic mechanisms.
TDI specific IgG antibodies have been found in subjects11, 24, 26, 30-32, TDI specific-IgG has been documented as a marker of exposure rather than of disease8. The presence of dNCO specific-IgE and -IgG have been widely investigated as diagnostic markers of occupational asthma in diisocyanate-exposed workers11, 21, 24, 29, 33-37.
Since effective assays are currently unavailable, a presumptive diagnosis of dNCO asthma is made from work history, report of work-related asthma-like symptoms and nonspecific airway reactivity to methacholine challenge.
In addition to asthma, irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD) can be produced by dermal exposure to dNCOs. Irritant contact dermatitis is the most common form of chemical induced dermatitis. It is a dose-dependent toxic/non-immunologically mediated effect associated with a chemical's ability to react with skin components and damage the skin. Allergic contact dermatitis is a T-lymphocyte mediated delayed (Type IV, DTH) hypersensitivity/immunological reaction.
Diagnosis of DTH is usually confirmed by clinical dermal patch testing. Currently there are three widely used standardized patch test: 1) Finn chamber, 2) True test and 3) Epiquick38. In these tests, the suspected sensitizing agent is dissolved/suspended in a solvent (usually petrolatum). A patch containing the diluted agent is applied, occluded onto skin, and read at 48, 72 and 96 hours. A patch test is interpreted based on observation of redness, itching and induration of skin at the site of the patch38, 39.
Biomonitoring of dNCOs involves either the measurement of specific antibody or of dNCO-conjugated biomolecules in blood or urine samples. Biomonitoring assays estimate total TDI exposure by converting TDI and its urinary metabolites to toluene diamine (TDA) by acid or base hydrolysis. A variety of analytical methods (e.g. chromatography) are used to determine the amount of TDA generated by laboratory hydrolysis40, 41. The detection of TDA in urine samples does not reflect the level of free TDA in the body, rather it estimates the combination of conjugated TDI derivatives and free-TDA42, 42, 43. This method does not distinguish between TDI and TDA exposure. Sabbioni et al. reported a dNCO biomarker assay employing mild base hydrolysis of hemoglobin from methylene diphenyl diisocyanate (MDI) exposed rats to yield the hydantoin from the MDI conjugated lysine of the N-terminal valine44.
There is a continuing need for compositions, such as monoclonal antibodies, and methods using the compositions, to isolate and characterize dNCO-conjugated proteins from dNCO-exposed humans and non-human animals. Compositions and methods are needed for use in detection of dNCO-exposure and related disease conditions in humans and non-human animals.